It is proposed to investigate three new problems concerning CNS TRH considered to be particularly relevant. The Principal Investigator has innovated methodologies for examining CNS TRH in a comprehensive schema embracing TRH biogenesis, TRH tissue content, TRH degradation, and TRH secretion. A simple and highly specific system has been developed for studying the biogenesis of TRH in vitro. Both hypothalamic and extrahypothalamic CNS tissues will be incubated in KRB buffer in vitro in the presence of 3H-L-proline (40 Ci/mMole), 10 minus to the 4th power M L-histidine, and 10 minus to the 4th power M L-glutamic acid. Following incubation, newly formed 3H-TRH will be isolated and purified by charcoal extraction, elution by acetic acid and sonication, and TRH-anti-TRH IgG Sepharose 4B affinity chromatography. Three new areas of inquiry will be explored: 1) The capacity of CNS structures containing TRH for TRH de novo biogenesis will be investigated, in conjunction with assessment of TRH tissue content, ongoing TRH degradation and TRH secretion; 2) Mechanisms operative in TRH biosynthesis will be studied in vitro by means of selected inhibitors of protein synthesis, including inhibitors of DNA transcription, RNA translation, non-ribosomal RNA, and enzymes. Potential larger TRH precursor peptides will be sought by means of specific immunoprecipitation of 3H-TRH-containing peptides with rabbit anti-TRH sera and SDS polyacrylamide gel electrophoretic resolution of 3H-peptides potentially involved in TRH biogenesis via enzymatic cleavage. The quantitative contribution of TRH formation by non-ribosomal mechanisms will be appraised by measurements of newly formed TRH (RIA) and 3H-TRH (affinity chromatography) vs time from pre-labelled tissues incubated with RNAase and cycloheximide and finally; 3) Neuropharmacological, hormonal, biological, and metabolic regulatory determinants of TRH biosynthesis, content, degradation and secretion will be examined in vitro.